Applications for surface immobilized biomolecules are widespread and include nucleic acid sequencing, gene expression profiling, analysis for single nucleotide polymorphisms (SNPs) and evaluation of hapten-antibody or ligand-target interactions. An important subset of these techniques involves immobilization of oligonucleotide probes that employ Watson-Crick hybridization in the interaction with target nucleic acids such as genomic DNA, RNA or cDNA prepared via Polymerase Chain Reaction (PCR) amplification of sample DNA. Current technologies often involve formatting oligonucleotide probes for such analyses into microarrays on glass slides, silicon chips or wafers, micro titer plates or other supports including polyacrylamide gel matrices.
A variety of methods exist for immobilizing biomolecules, including non-covalent (hydrophobic or ionic interactions) as well as covalent methods. A number of these methods are summarized in Table 1. Methods involving covalent attachment are generally considered preferable, as more stringent conditions may be applied to the immobilized system for the reduction of non-specific ionic or hydrophobic associations (which raise background signal) without concern for the loss of the probe from the surface. Commonly employed covalent methods include condensation of amines with activated carbonyl groups on the surface, such as activated carboxylic acid esters, carbonates or isocyanates or isothiocyanates. Additionally, amine groups can be condensed with aldehydes under reductive amination conditions to afford secondary amine linkages between the surface and the biomolecule. Furthermore, amines can be condensed with electron deficient heterocycles via nucleophilic aromatic substitution as well as epoxide opening.
Cycloaddition reactions can be defined as any reaction between two (or more) moieties (either intra or intermolecular) where the orbitals of the reacting atoms form a cyclic array as the reaction progresses (typically in a concerted fashion although intermediates may be involved) along the reaction coordinate leading to a product. The orbitals involved in this class of reactions are typically π systems although certain σ orbitals can also be involved. The number of electrons associated with this type of reaction are of two types: 4n+2 and 4n, where n=0, 1, 2, 3, 4, etc. Typical examples of cycloaddition reactions include Diels-Alder cycloaddition reactions, 1,3-dipolar cycloadditions and [2+2] cycloadditions.
The Diels-Alder reaction, by far the most studied cycloaddition, is the cycloaddition reaction between a conjugated diene and an unsaturated molecule to form a cyclic compound with the π-electrons being used to form the new σ-bonds. The Diels-Alder reaction is an example of [4+2] cycloaddition reaction, as it involves a system of 4π-electrons (the diene) and a system of 2π-(the dienophile). The reaction can be made to occur very rapidly, under mild conditions, and for a wide variety of reactants. The Diels-Alder reaction is broad in scope and is well known to those knowledgeable in the art. A review of the Diels-Alder reaction can be found in “Advanced Organic Chemistry” (March, J., ed.) 839-852 (1992) John Wiley & Sons, NY, which is incorporated herein by reference.
It has been discovered that the rate of Diels-Alder cycloaddition reactions is enhanced in aqueous solvents. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816). (A similar effect is also seen with 1,3-dipolar cycloaddition reactions (Engberts (1995) Tetrahedron Lett. 36:5389). This enhancement is presumably due to the hydrophobicity of the diene and dienophile reactants. (Breslow (1991) Acc. Chem. Res. 24:159). This effect extends to intramolecular Diels-Alder reactions. (Blokzijl et al. (1991) J. Am. Chem. Soc. 113:4241). Not only is the reaction rate accelerated in water, but several examples of an increased endo/exo product ratio are also reported. (Breslow and Maitra (1984) Tetrahedron Lett. 25:1239; Lubineau et al. (1990) J. Chem. Soc. Perkin Trans. I, 3011; Grieco et al. (1983) Tetrahedron Lett. 24:1897). Salts which increase the hydrophobic effect in water, such as lithium chloride (Breslow et al. (1983) Tetrahedron Lett. 24:1901) and also monovalent phosphates (Pai and Smith (1995) J. Org. Chem. 60:3731) have been observed to further accelerate the rate of [4+2] cycloadditions.
In U.S. application Ser. No. 09/051,449, filed Apr. 6, 1998; Ser. No. 08/843,820, filed Apr. 21, 1997 and Ser. No. 09/402,430, filed Oct. 7, 1999; each entitled “Method for Solution Phase Synthesis of Oligonucleotides,” the Diels-Alder cycloaddition reaction is shown to be an ideal method for anchoring oligonucleotides onto resins. Resins derivatized with a diene or dienophile are reacted with an oligonucleotide derivatized with a dienophile or diene, respectively, to yield the Diels-Alder cycloaddition product. In particular, Diels-Alder reactions between oligonucleotides derivatized with a diene and polymeric resins derivatized with maleimide groups and with phenyl-triazoline-diones (PTAD) are described. The resulting resins can be used as affinity chromatography resins.
U.S. application Ser. No. 09/341,337, filed Jul. 7, 1999, entitled “Bioconjugation of Macromolecules,” illustrates that cycloaddition reactions in general, such as the Diels-Alder reaction and 1,3-dipolar cycloaddition reactions, are an ideal replacement for current methods of conjugating macromolecules with other molecular moieties. The Diels-Alder reaction, in particular, is an ideal method for covalently linking large water soluble macromolecules with other compounds as the reaction rate is accelerated in water and can be run at neutral pH. (Rideout and Breslow (1980) J. Am. Chem. Soc. 102:7816). Additionally, the nature of the reaction allows post-synthetic modification of the hydrophilic macromolecule without excess reagent or hydrolysis of the reagent. With respect to conjugation to oligonucleotides, this technology has been aided by the ability to efficiently synthesize 2′-O-diene-nucleosides, which allows the conjugation site to be varied throughout the oligonucleotide or the option of having multiple conjugation sites.
The present invention describes a method for immobilizing molecules, particularly biomolecules, to a support using the cycloaddition bioconjugation method. Immobilization of biomolecules via cycloaddition, particularly Diels-Alder reactions, offers the following major advantages over conventional methods (cf. Table 1): cycloaddition reactions establish a covalent and stable linkage between the linked compounds; the reaction proceeds with high chemoselectivity; functional groups of biomolecules do not interfere with the cycloaddition reaction; the cycloaddition reaction is orthogonal to other immobilization/labeling protocols, thus two-fold reactions are possible in one reaction mixture; in contrast to general techniques in organic synthesis, as discussed above, Diels-Alder reactions, can be carried out in aqueous phase, the Diels-Alder reaction is tremendously accelerated in water and is very fast at room temperature or slightly below; the cycloaddition reaction proceeds under neutral conditions in a one-step procedure; no by-products are formed during the reaction; no activators or additives are necessary to run the reaction and the moieties involved in the reaction (dienes and dienophiles) are stable under various reaction conditions employed for conjugation or immobilization of biomolecules
Note, that throughout this application various citations are provided. Each citation is specifically incorporated herein by reference in its entirety.